Uncoupling of Ca2+ sparks from BK channels in cerebral arteries underlies hypoperfusion in hypertension-induced vascular dementia

Significance There are no treatments available for vascular dementia, the second-most common dementia syndrome, and patients decline rapidly after diagnosis. This syndrome is primarily due to hypertension and is associated with reduced cerebral blood flow (CBF). To explore this, we studied mechanisms of vascular dysfunction in pial arteries from hypertensive mice. These mice exhibit reduced CBF, hyperconstricted pial arteries, and behavior approximating human vascular dementia. Using myography, electrophysiology, and Ca2+ imaging, we found that the increased constriction was due to separation of the sarcoplasmic reticulum from the plasma membrane in vascular smooth muscle cells, which prevented vasodilatory Ca2+ signals from activating large-conductance K+ channels. We propose that restoring this coupling could improve CBF and slow disease progression.


Animal studies
All procedures used in this study were approved by The University of Manchester Animal Welfare Ethical Review Board and were conducted in accordance with UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986. Spontaneously hypertensive mice (BPH/2) and their normotensive counterparts (BPN/3) were originally sourced from Jackson Labs, Florida, before breeding was undertaken at the University of Manchester. Eight-month-old male BPH/2 and age-matched BPN/3 mice were used throughout this study. Mice had free access to food and water and were housed in pathogen-free conditions under a 12 h day/night cycle.

Laser-Doppler blood flow measurements
Mice were anesthetized with 4% isoflurane (Abbott, Berkshire, UK) and secured in a stereotaxic frame (World Precision Instruments, USA) positioned under a Moor FLPI2 Laser Speckle Imager (Moor Instruments, UK). Isoflurane anesthesia was maintained at 1.5% throughout imaging, and body temperature was maintained at 37°C using an electric heating pad and monitored using an anal thermometer. The skull was exposed by dissecting the scalp along the midline, and the region was cleared for imaging by securing the skin on top of the skull using surgical clips. An ultrasound gel was applied to the mouse skull and a glass coverslip was mounted on top to improve imaging quality. Laser speckle cranial imaging was conducted for 10 min with a frame rate of 1500/min. Following imaging, mice were euthanized by cervical dislocation. A region of interest was drawn around the same middle cerebral artery branch in each image using Moor FLPI2 software (Axminster, UK), and the median flux was averaged over the last 5 min of the recording. (Readings fluctuated during the first 5 min before animals reached a steady state of anesthesia.)

Behavioral assessments
Behavior was assessed during the light phase (7 am to 7 pm) unless otherwise stated. After assigning to cages according to group, mice were individually handled on three consecutive days before testing. All tests were performed after a minimum of 1 h acclimatization to the experimental room. Each mouse received an individual code, and experimenters were blinded for analysis of behavioral tests, except for manual video scoring for the novel object-recognition test. In the latter, the genotype of the mice could be distinguished due to their different fur coloring-white for BPH/2 and brown for BPN/3 animals.
Nesting: Spontaneous, innate nesting behavior was assessed by first placing mice in individual cages containing 20 g of Sizzle-Nest material (Datesand LTD, Manchester, UK) 1 h before the dark phase and then leaving them overnight with free access to food and water. Nests were scored the next day during the light cycle by two independent assessors. Scoring was performed as previously described (1). Briefly, nests were scored as follows: 0, material not manipulated; 1, no clear nest site (majority of nesting material not manipulated to one cage quadrant); 2, nest present but flat; 3, nest present with raised walls <30 mm in height; 4, nest present with walls 31-49 mm in height; and 5, nest present with walls > 50 mm in height.
Burrowing: As an additional assessment of well-being, two burrowing tests were conducted at least 48 h apart. In these tests, mice were singly housed for 2 h, and 150 g of standard diet food pellets (Envigo, UK) were added inside custom-made burrowing tubes (200-mm lengths of 68 -mm diameter PVC downpipe) as previously described (2,3). Thereafter, the quantity of food pellets burrowed was recorded and mice were returned to their home cages.
Open field: For open-field tests, mice were individually placed in a square Perspex arena (450 × 450 × 200 mm) and allowed to explore freely for 5 min. Video recordings were obtained from a camera suspended above the arena and automatically analyzed using ANY-Maze v7.10 software (Stoelting, USA). The following parameters were automatically calculated: distance travelled, number of entries and time spent in the center zone, and freezing time (1-s threshold). Fecal pellet droppings were also counted at the end of the test.
Novel object recognition: The novel object recognition (NOR) test was used to assess short-term memory. A round arena, 30 cm in diameter, was fitted with two identical objects functioning as familiar objects for habituation and acquisition phases. A novel object was randomly assigned for the retention phase. Objects were created with Lego pieces and cleaned between each use. After habituating to the arena as a cage-based group, mice were subject to a 5-min acquisition period, followed by a 5-min retention test 4 h later. Active exploration was defined as touching, sniffing, or exploring the objects. Each phase was video recorded from above, and object-interaction time was manually timed. Recognition index was calculated using the formula, TN/(TN+TF), where TN is the time spent exploring the novel object and TF is the time spent exploring the familiar object, as previously described (4).

Pressurized arteries
For pressure myography, imaging and electrophysiology experiments, mice were euthanized by overdose of CO2 followed by exsanguination. The brain was removed and kept in ice-cold physiological saline solution (Mg-PSS; 140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 10 mM HEPES and 10 mM glucose; pH adjusted to 7.4 with 1 M NaOH) before conducting experiments. Cerebral pial resistance arteries were dissected from the brain and stored in Mg-PSS on ice.
Cerebral pial (posterior cerebral or superior cerebellar) arteries were dissected from the brain and stored in chilled Mg-PSS. Arterial segments were mounted on glass pipettes of a similar size in an arteriograph chamber (Living Systems Instrumentation, VT). Arteries were allowed to equilibrate for 15 min in pre-warmed (37°C) physiological saline solution (PSS; 125 mM NaCl, 3 mM KCl, 26 mM NaHCO3, 1.25 mM NaH2PO4·H2O, 1 mM MgCl2, 4 mM glucose and 2 mM CaCl2, bubbled with 5% CO2 in biological air) before being pressurized to 60 mmHg. Vessels were allowed to develop myogenic tone before using in either pressure-response or paxilline experiments. Luminal diameter was continuously measured throughout the experiment using a camera and edge-detection software (IonOptix). Percentage constriction was calculated as the degree of change relative to aerated with 5% CO2/21% O2 (balance N2) and warmed to 37°C. After a 20-min equilibration period at 5 mmHg, arteries were pressurized to 60 mmHg. Thereafter, Ca 2+ events were imaged (excitation wavelength, 488 nm; fluorescence emission collected above 510 nm) using a highspeed spinning-disc confocal microscope (Nikon Eclipse TE-2000U). Images (512 × 512 pixels; field of view, 131 × 131 μm) were recorded every 18.9 ms (53 fps) using a 60× water-immersion objective (final magnification, 600×; NA1.2).
Ca 2+ events were analyzed as previously described (5). In brief, videos of Ca 2+ events were imported into custom-written software (Volumetry G9e; G.W.H) before being motion-stabilized and normalized with respect to background intensity. Because basal fluorescence intensity varies across VSMCs, this analysis records basal fluorescence at quiescence (SDq) and gives a Z-score (Zscr) to increases from this value. A threshold is applied to the image such that only sufficiently large increases in Zscr are defined as Ca 2+ events. A range of quantitative metrics were extracted from the videos, including duration, size and direction of spread, and maximum intensity (Zscr).
Using these data, we defined Ca 2+ sparks as signals with a duration of less than 0.4 seconds and a spatial spread less than 5 μM, and Ca 2+ waves as events with a duration of 0.5-2 s and a spatial spread covering more than 50% of the cell.

VSMC isolation
VSMCs were isolated by digesting cerebral pial arteries in Mg-PSS supplemented with papain (1.0 mg/ml; Worthington Biochemical, NJ, USA), dithioerythritol (1 mg/ml) and BSA (10 mg/ml) at 37°C for 12 min, washed three times with Mg-PSS, and then incubated a second time for 14 min at 37°C in type II collagenase (1.0 mg/ml; Worthington). VSMCs were then liberated by triturating digested arteries, stored in ice-cold Mg-PSS, and studied within 6 h.

Electrophysiology
Currents were recorded using an AxoPatch 200B amplifier equipped with an Axon CV 203BU headstage (Molecular Devices), filtered at 1 kHz, digitized at 40 kHz, and stored for subsequent analysis. Clampex and Clampfit (version 10.2; Molecular Devices) were used for data acquisition and analysis, respectively. All recordings were performed at room temperature (~22°C). VSMCs were transferred to a recording chamber and allowed to adhere to glass coverslips for 10 min at

Live-cell imaging of the SR and PM
SMCs isolated from BPN/3 and BPH/2 pial arteries were allowed to adhere to glass-bottom 35-mm dishes (Corning; Fisher Scientific) for 1 h on ice. The SR and PM were labeled using a protocol that we previously described (6,7). VSMCs were treated with ER-Tracker Green (

Proximity ligation assay
SMCs isolated from BPN/3 and BPH/2 pial arteries were allowed to adhere to 22 x 22 mm coverslips for 1 h on ice, then fixed with 2% paraformaldehyde for 15 min at room temperature.
Thereafter, cells were washed with 1x phosphate-buffered saline (PBS; Sigma-Aldrich), permeabilized with 0.1% Triton X-100 (Sigma Aldrich) for 10 min, and blocked with 50% SEA Block     waves, defined as events that occupied more than 50% of the cell and lasted between 0.5 and 2 seconds (N = 7 vessels from 7 normotensive mice and 10 vessels from 10 hypertensive mice; unpaired t-test).

Legends for Movies
Movie S1 Animated representation of a cerebral artery SMCs isolated from a normotensive mouse, labeled with live-cell membrane dyes and imaged using deconvolved confocal microscopy. Images were reconstructed and rendered to show the PM (red), SR (cyan), and peripheral coupling sites (PCS; yellow).

Movie S2
Animated representation of a cerebral artery SMCs isolated from a hypertensive mouse, labeled with live-cell membrane dyes and imaged using deconvolved confocal microscopy. Images were reconstructed and rendered to show the PM (red), SR (cyan), and peripheral coupling sites (PCS; yellow).

Movie S3
Animated representation of a cerebral artery SMCs isolated from a normotensive mouse, showing positive proximity ligation assay puncta (magenta).
Movie S4 Animated representation of a cerebral artery SMCs isolated from a hypertensive mouse, showing positive proximity ligation assay puncta (magenta).